The Ferredoxin-Binding Site of Ferredoxin:Nitrite Oxidoreductase (Differential Chemical Modification of the Free Enzyme and Its Complex with Ferredoxin)

Abstract

Fd:nitrite oxidoreductases (EC 1.7.7.1, hereafter referred to as nitrite reductase) catalyze the six-electron reduction of nitrite to ammonia, using reduced Fd as the electron donor (Knaff, 1996). Fd-dependent nitrite reductases have been isolated and characterized from a number of higher plants, algae, and cyanobacteria and have been shown to be monomeric proteins with molecular masses near 63 kD, which contain a single [4Fe-4S] cluster and a single siroheme (which serves as the binding site for nitrite) as prosthetic groups (Knaff, 1996). The spinach (Spinacea oleracea) leaf enzyme, which is localized in the chloroplast stromal space, has been particularly well characterized (Knaff, 1996), and its complete amino acid sequence has been deduced from a cDNA sequence and N-terminal amino acid sequencing of the mature form of the protein (Back et al

Proteomics Uncovers Proteins Interacting Electrostatically with Thioredoxin in Chloroplasts

“…Ferredoxin is known to form a strong interaction with different partner proteins to allow efficient electron transfer from PS I to the final acceptor -that is, NADP (ferredoxin-NADP reductase, or FNR), nitrite (nitrite reductase), sulfite (sulfite reductase), α-ketoglutarate (glutamate synthase) and thioredoxin (FTR) (Droux et al 1987;Dose et al 1997;GarciaSanchez et al 1997;Akashi et al 1999). When bound to the thylakoid membrane, such complexes efficiently transfer electrons form PS I to their substrates (Knaff and Hirasawa 1991).…”

Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism

“…Work on the X-ray crystal structure of spinach Fdglutamate synthase (J.P. Allen, A. Artigas Camara, M. Hirasawa, D.B. Knaff, unpublished observations) has not yet progressed to the point where it can be stated unambiguously that a similar loop is present in the spinach enzyme and the large size of spinach Fd-glutamate synthase has made it impossible to use peptide-mapping techniques, of the type used successfully with FNR (Jelesarov et al 1993) and ferredoxin-dependent nitrite reductase (Dose et al 1997), to identify specific lysine and arginine residues that may be involved in binding ferredoxin.…”

A Conserved Histidine-Aspartate Pair Is Required for Exovinyl Reduction of Biliverdin by a Cyanobacterial Phycocyanobilin:Ferredoxin Oxidoreductase

“…The importance of a positively charged "docking" surface for reduced Fd has been reported for other Fd-dependent enzymes, e.g. FNR, nitrite reductase, and sulfite reductase (11,12,14,16 (5). In that study, we envisaged four proton-donating residues, D 1 -D 4 , to mediate these electron/proton transfer steps (Fig.…”

“…Replacement of this conserved lysine (Lys 218 ) with glutamic acid failed to abolish the activity of the enzyme, although the 28EK1 mutant retained only 20% of wild type activity (Table 1). In view of this observation, the results from TNBS modification experiments can better be rationalized by the participation of TNBS-modifiable lysine residue(s) in the electrostatic interaction between PcyA and reduced Fd, an interaction critical for the activity of other Fd-dependent enzymes (11)(12)(13)(14)(15). We believe that it is less likely that Lys 218 plays a direct proton-donating role for PcyA-mediated bilin reduction.…”